Mitochondria are the intracellular organelles responsible for energy production in eukaryotic cells, and are unique in that they contain their own DNA (mtDNA), which encodes genes important for mitochondrial function. While it has been well-established that nuclear DNA has an increased overall burden of mutations in cancers, we recently discovered that the frequency of random mutations in mtDNA is decreased in human tumors relative to healthy tissues. Furthermore, these novel findings provide the framework for the proposed research. We have since demonstrated that the lower burden of mtDNA mutations in tumor tissue is coupled to a decrease in oxidative phosphorylation (OXPHOS) and, by extension, a reduction in reactive oxygen species (ROS)-mediated mtDNA damage. These novel findings provide the impetus for the proposed research, as they suggest that, unlike in nuclear DNA, an increased rate of mtDNA mutagenesis does not facilitate a cancer?s development; rather, it may hinder it. As such, under the direction of the proposed Specific Aims we expect to delineate the mechanisms underlying mitochondrial mutagenesis in normal and tumor cells, and exploit these for use in the clinic. For the first Aim, we propose to delineate the relationships and among metabolism, ROS, mtDNA mutagenesis, and apoptotic priming by testing the hypothesis that mtDNA mutagenesis mediates apoptotic response. Secondly, we will establish whether mtDNA mutagenesis is predictive of treatment response and thus can serve as a specific, prognostic biomarker of pathological response (pCR) to neoadjuvant treatment in locally advanced breast cancer (LABC). Lastly, we will determine if mitochondrial- targeted cancer therapeutics, focused on directly increasing OXPHOS, will promote apoptotic response and sensitize cancer cells to chemotherapeutically-induced apoptosis. Successful completion of the proposed project Aims will: (1) Establish a cause and effect relationship between glucose metabolism and apoptotic resistance; (2) Advance understanding of relationships between mtDNA mutagenesis, cell metabolism, and cancer; (3) Determine the utility of mitochondrial mutagenesis as a predictive biomarker of treatment response, early relapse, and death; (4) Explore the utility of a novel chemotherapeutic strategy that increases mitochondrial respiration; and (5) ultimately improve cancer prognostication and treatment, thereby improving patient outcomes and quality of life.